Infectious diseases remain a major health problem and cause of death worldwide. The identification and localization of sites of infection in patients is a critical step in their clinical management, particularly when localized symptoms of the infection are not present. The prompt identification of infections can be cured with proper treatment, while delays can result in mortality. Anatomic imaging using ultrasound or computed tomography can be used for identification if local symptoms are present, however, they cannot differentiate between an infection and inflammation. Whole body scans in nuclear medicine imaging would be advantageous for imaging when there are no local symptoms of infection. There are a variety of radiopharmaceuticals that are used to detect infection and inflammation; however, it has been shown that the majority of these probes cannot distinguish between infection and inflammation. Therefore, the development of an imaging agent that is specific for detection of bacterial infections in a variety of clinical contexts would be highly significant. Soe progress has been made on the development of probes for the specific detection of viral, fungal, and bacterial infections. The use of radiolabeled antibiotics such as ciprofloxacin, kanamycin, and sulphanilamide for the detection of bacterial infections has been investigated, but there is concern about their specificity. The goal of this study is to develop a PET imaging agent based on a novel class of antimicrobial agents for the specific detection of bacterial infections. These novel antimicrobial agents are cationic steroid compounds, designed to mimic the morphology of endogenous antimicrobial peptides and have been shown to be effective against both Gram-positive and Gram-negative bacteria. These compounds display high and selective affinity for bacterial membranes over eukaryotic cells. In these studies, we will modify the cationic steroid antibiotics (CSAs) with the 1, 4, 7-triazacyclononane-1, 4, 7-triacetic acid (NOTA) chelator such that they can be radiolabeled with the positron-emitter, 64Cu. These conjugates will be evaluated to ensure that they maintain their activity against bacteria and then radiolabeled with 64Cu to determine uptake and affinity in bacterial cultures. The resulting radiolabeled conjugates will then be evaluated in a lung infection model in sterile inflammation to determine the specificity of the agent. The specific aims of the proposal are: 1.) Synthesize and evaluate CSAs that have been conjugated to NOTA and 2.) Evaluate 64Cu-NOTA-CSA conjugates in a lung infection model.